Human cells encode proteins that naturally suppress HIV-1 infection. One of these proteins, APOBEC3G (A3G), has anti-HIV-1 activity that can be neutralized by a virally encoded protein, Vif. A3G is a cytidine deaminase that induces the modification of cytosines to uracils in newly synthesized minus- strand viral DNA, resulting in non-functional viruses in the absence of Vif. When Vif is present, it prevents the packaging of A3G, allowing the production of infectious virions. Over the last several years, with funding from our current grant, we have characterized a complex series of proteins, including Cul5, Elongin B, Elongin C, and Rbx, which combine with Vif to form an E3 ubiquitin ligase complex that enables HIV to bypass the APOBEC3 restriction and replicate. Characterizing these proteins is critical to understanding how HIV-1 Vif overcomes host defenses. Recently, we have demonstrated that a novel cellular factor, core binding factor beta (CBF?), is another key regulator of Vif's ability to evade the host's intrinsic APOBEC3-mediated defense system. Our data indicate that CBF? apparently operates via a novel mechanism to affect Cul5-E3 ubiquitin ligase assembly. Preventing the interaction between Vif and CBF? represents an attractive pharmacologic target in HIV-1 infection. In this application, we propose to further characterize the role of CBF? and its known co-factors in the functioning of HIV-1 Vif. In particular, we propose the following Specific Aims: (1). To characterize the role of CBF? in the formation of the Vif- Cul5-ElonginB-ElonginC E3 ubiquitin ligase and the evasion of host APOBEC3 restriction. Experiments in this Aim are designed to investigate the role of CBF? in the regulation of Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase activity and the suppression of host anti-viral APOBEC3 proteins. (2). To examine the role of known CBF? co-factors in HIV-1 Vif function. The role of various RUNX proteins, filaminA, charged amino acid rich leucine zipper-1 (Crl-1), and HIPK2, in HIV-1 Vif function will be examined. These proteins could be enhancers of Vif function or competitive inhibitors of Vif. (3). To study the molecular interaction between HIV-1 Vif and CBF?. A complete understanding of Vif- CBF? activity and the rational design of inhibitors of this critical target require a clearer picture of the protein-protein interactions that occur within the Vif- CBF? complex. The studies proposed in this Aim will lay the groundwork for future research in this field. Information regarding the molecular interactions between Vif and CBF? may also assist us in the structural characterization of Vif molecules.